Epilepsy is characterized by two pathological electrographic phenotypes: seizures and interictal spikes. Seizures are relatively rare sustained elevations in brain activity or synchronization that often produce a loss of consciousness in patients suffering from epilepsy. Interictal spikes are much more frequent (on the order of 1 per minute), brief (~200ms), and have no behavioral phenotype (patient is typically not aware of interictal spikes). It is well-established that epileptic networks commonly generate both types of discharge and mounting evidence suggests that there is a direct link between interictal spikes and seizure onset. This proposal aims to dissect the basic mechanisms of interictal spike generation, which will offer new insight into the dynamics of epileptic neural networks (and, in turn, inform development of novel therapeutics). The proposed project will investigate the origins of interictal spikes using an experimentally accessible preparation, the organotypic slice culture (which spontaneously develops interictal spikes and seizures during its first weeks in culture), and a custom microscope (the ?Incuscope?) specially designed to record and manipulate activity with single-cell resolution, across the entire epileptic network. Imaging the entire epileptic network guarantees that epileptiform activity observed is not driven by external input. Furthermore, the Incuscope is built inside of a tissue culture incubator, enabling continuous, month-long imaging as epileptic activity emerges and evolves. Findings will be validated in intact animals using endomicroscopy-based in vivo imaging of interictal spikes. The primary goals of this project are as follows: 1) Identify subpopulations of early-firing cells during interictal spikes. 2) Optically stimulate early-firing cells to characterize the degree to which their activation is sufficient to initiate spikes. 3) Optically inhibit early-firing cells to characterize the degree to which their activation is necessary to initiate spikes. 4) Repeat experiments 1-3 in long-term (multi- week) recordings to characterize the stability of early-firing cells and correlate observed changes with seizure onset and seizure burden. 5) Repeat experiment 1 in awake mice, using endomicroscopy and the intrahippocampal kainate model of epilepsy, to test whether the same population of cells is involved in generating interictal spikes in vivo.